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Genes with similar expression patterns in a set of diverse samples may be considered coexpressed. Human Gene Coexpression Analysis 2.0 (HGCA2.0) is a webtool which studies the global coexpression landscape of human genes. The website is based on the hierarchical clustering of 55,431 Homo sapiens genes based on a large-scale coexpression analysis of 3500 GTEx bulk RNA-Seq samples of healthy individuals, which were selected as the best representative samples of each tissue type. HGCA2.0 presents subclades of coexpressed genes to a gene of interest, and performs various built-in gene term enrichment analyses on the coexpressed genes, including gene ontologies, biological pathways, protein families, and diseases, while also being unique in revealing enriched transcription factors driving coexpression. HGCA2.0 has been successful in identifying not only genes with ubiquitous expression patterns, but also tissue-specific genes. Benchmarking showed that HGCA2.0 belongs to the top performing coexpression webtools, as shown by STRING analysis. HGCA2.0 creates working hypotheses for the discovery of gene partners or common biological processes that can be experimentally validated. It offers a simple and intuitive website design and user interface, as well as an API endpoint.

Background: Eukaryotic genomes undergo pervasive transcription, leading to the production of many types of stable and unstable RNAs. Transcription is not restricted to regions with annotated gene features but includes almost any genomic context. Currently, the source and function of most RNAs originating from intergenic regions in the human genome remain unclear., Results: We hypothesize that many intergenic RNAs can be ascribed to the presence of as-yet unannotated genes or the "fuzzy" transcription of known genes that extends beyond the annotated boundaries. To elucidate the contributions of these two sources, we assemble a dataset of more than 2.5 billion publicly available RNA-seq reads across 5 human cell lines and multiple cellular compartments to annotate transcriptional units in the human genome. About 80% of transcripts from unannotated intergenic regions can be attributed to the fuzzy transcription of existing genes; the remaining transcripts originate mainly from putative long non-coding RNA loci that are rarely spliced. We validate the transcriptional activity of these intergenic RNAs using independent measurements, including transcriptional start sites, chromatin signatures, and genomic occupancies of RNA polymerase II in various phosphorylation states. We also analyze the nuclear localization and sensitivities of intergenic transcripts to nucleases to illustrate that they tend to be rapidly degraded either on-chromatin by XRN2 or off-chromatin by the exosome., Conclusions: We provide a curated atlas of intergenic RNAs that distinguishes between alternative processing of well-annotated genes from independent transcriptional units based on the combined analysis of chromatin signatures, nuclear RNA localization, and degradation pathways.

Once believed to be unique features of neoplasia, chimeric RNAs are now being discovered in normal physiology. We speculated that some chimeric RNAs may be functional precursors of genes, and that forming chimeric RNA at the transcriptional level may be a 'trial' mechanism before the functional element is fixed into the genome. Supporting this idea, we identified a chimeric RNA, HNRNPA1L2-SUGT1 (H-S) , whose sequence is highly similar to that of a 'pseudogene' MRPS31P5 . Sequence analysis revealed that MRPS31P5 transcript is more similar to H-S chimeric RNA than its 'parent' gene, MRPS31 . Evolutionarily, H-S precedes MRPS31P5 , as it can be detected bioinformatically and experimentally in marmosets, which do not yet possess MRPS31P5 in their genome. Conversely, H-S is minimally expressed in humans, while instead, MRPS31P5 is abundantly expressed. Silencing H-S in marmoset cells resulted in similar phenotype as silencing MRPS31P5 in human cells. In addition, whole transcriptome analysis and candidate downstream target validation revealed common signalling pathways shared by the two transcripts. Interestingly, H-S failed to rescue the phenotype caused by silencing MPRS31P5 in human and rhesus cells, whereas MRPS31P5 can at least partially rescue the phenotype caused by silencing H-S in marmoset cells, suggesting that MRPS31P5 may have further evolved into a distinct entity. Thus, multiple lines of evidence support that MRPS31P5 is not truly a pseudogene of MRPS31 , but a likely functional descendent of H-S chimera. Instead being a gene fusion product, H-S is a product of cis-splicing between adjacent genes, while MRPS31P5 is likely produced by genome rearrangement.

The frequency by which genes are studied correlates with the prior knowledge accumulated about them. This leads to an imbalance in research attention where some genes are highly investigated while others are ignored. Geneshot is a search engine developed to illuminate this gap and to promote attention to the under-studied genome. Through a simple web interface, Geneshot enables researchers to enter arbitrary search terms, to receive ranked lists of genes relevant to the search terms. Returned ranked gene lists contain genes that were previously published in association with the search terms, as well as genes predicted to be associated with the terms based on data integration from multiple sources. The search results are presented with interactive visualizations. To predict gene function, Geneshot utilizes gene-gene similarity matrices from processed RNA-seq data, or from gene-gene co-occurrence data obtained from multiple sources. In addition, Geneshot can be used to analyze the novelty of gene sets and augment gene sets with additional relevant genes. The Geneshot web-server and API are freely and openly available from https://amp.pharm.mssm.edu/geneshot., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Comparative epigenomic analysis across multiple genes presents a bottleneck for bench biologists working with NGS data. Despite the development of standardized peak analysis algorithms, the identification of novel epigenetic patterns and their visualization across gene subsets remains a challenge., Results: We developed a fast and interactive web app, C-State (Chromatin-State), to query and plot chromatin landscapes across multiple loci and cell types. C-State has an interactive, JavaScript-based graphical user interface and runs locally in modern web browsers that are pre-installed on all computers, thus eliminating the need for cumbersome data transfer, pre-processing and prior programming knowledge., Conclusions: C-State is unique in its ability to extract and analyze multi-gene epigenetic information. It allows for powerful GUI-based pattern searching and visualization. We include a case study to demonstrate its potential for identifying user-defined epigenetic trends in context of gene expression profiles.

Two strategies are often adopted for enrichment analysis of pathways: the analysis of all differentially expressed (DE) genes together or the analysis of up- and downregulated genes separately. However, few studies have examined the rationales of these enrichment analysis strategies. Using both microarray and RNA-seq data, we show that gene pairs with functional links in pathways tended to have positively correlated expression levels, which could result in an imbalance between the up- and downregulated genes in particular pathways. We then show that the imbalance could greatly reduce the statistical power for finding disease-associated pathways through the analysis of all-DE genes. Further, using gene expression profiles from five types of tumours, we illustrate that the separate analysis of up- and downregulated genes could identify more pathways that are really pertinent to phenotypic difference. In conclusion, analysing up- and downregulated genes separately is more powerful than analysing all of the DE genes together.

It is known that chronic social stress leads to immunity disorders in humans and experimental animals. It has been shown that the effect of stress is also manifested in changes in the level of expression of genes involved in the functioning of various physiological systems in the brain of mice, in particular, in the hypothalamus. It was noted that in stressed animals, genes involved in the processes of carcinogenesis and apoptosis change their expression, and in animals without signs of developing a malignant process, but under conditions conducive to tumor growth. In this regard, we used the RNA-seq method to study the expression of cytokine response genes in the hypothalamus of male mice under the influence of chronic social stress caused by repeated experience of defeats in intermale confrontations, compared with control individuals. Multidirectional changes in the expression of cytokine genes, their receptors and genes performing a regulatory function were detected (IL17d, IL18, IL33, Csf1r, Csf2ra, IL11ra1, IL13ra1, IL2ra, IL3ra, IL5ra, Lifr, Cish, IL4i1, Irf1, Irf5, Irf9, Jak2, Socs3, Stat3, Tgfb1, Tlr3). Thus, it has been shown that changes in the cytokine response in the brain under the influence of stress occur at the level of changes in gene expression. In this case, we should not talk about the activation of the system or a decrease in its activity, but about the disruption of its functioning. Next, we analyzed the correlations between the level of expression of genes of the cytokine system and the main genes of carcinogenesis and apoptosis that we studied earlier (Akt1, Bag6, Foxp4, Mapk3, Mapk8, Nol3, Pdcd10, Xiap). The Akt1, Jak2, Stat3 genes were identified, for which the maximum number of correlations was found, moreover, negative correlations were most characteristic of Jak2, and positive correlations were most characteristic of Stat3 and Akt1. In addition, protein-protein interactions between genes of carcinogenesis and apoptosis and genes of the cytokine system were analyzed using the String database in mice under chronic social stress. It was confirmed the key role of these genes in the development of dysfunction of cytokines in the brain.

Litter size is a key indicator of production performance in livestock. However, its genetic basis in goats remains poorly understood. In this work, a genome‐wide selection sweep analysis (GWSA) on 100 published goat genomes with different litter rates was performed for the first time to identify candidate genes related to kidding rate. This analysis was combined with the public RNA‐sequencing data of ovary tissues (follicular phase) from high‐ and low‐yielding goats. A total of 2278 genes were identified by GWSA. Most of these genes were enriched in signaling pathways related to ovarian follicle development and hormone secretion. Moreover, 208 differentially expressed genes between groups were obtained from the ovaries of goats with different litter sizes. These genes were substantially enriched in the cholesterol and steroid synthesis signaling pathways. Meanwhile, the weighted gene co‐expression network was used to perform modular analysis of differentially expressed genes. The results showed that seven modules were reconstructed, of which one module showed a very strong correlation with litter size (r = −0.51 and p‐value <0.001). There were 51 genes in this module, and 39 hub genes were screened by Pearson's correlation coefficient between core genes > 0.4, correlation coefficient between module members > 0.80 and intra‐module connectivity ≥5. Finally, based on the results of GWSA and hub gene Venn analysis, seven key genes (ACSS2, HECW2, KDR, LHCGR, NAMPT, PTGFR and TFPI) were found to be associated with steroid synthesis and follicle growth development. This work contributes to understanding of the genetic basis of goat litter size and provides theoretical support for goat molecular breeding. [ABSTRACT FROM AUTHOR]

Background: Real-time quantitative PCR (RT-qPCR) is one of the most widely used gene expression analyses for validating RNA-seq data. This technique requires reference genes that are stable and highly expressed, at least across the different biological conditions present in the transcriptome. Reference and variable candidate gene selection is often neglected, leading to misinterpretation of the results. Results: We developed a software named "Gene Selector for Validation" (GSV), which identifies the best reference and variable candidate genes for validation within a quantitative transcriptome. This tool also filters the candidate genes concerning the RT-qPCR assay detection limit. GSV was compared with other software using synthetic datasets and performed better, removing stable low-expression genes from the reference candidate list and creating the variable-expression validation list. GSV software was used on a real case, an Aedes aegypti transcriptome. The top GSV reference candidate genes were selected for RT-qPCR analysis, confirming that eiF1A and eiF3j were the most stable genes tested. The tool also confirmed that traditional mosquito reference genes were less stable in the analyzed samples, highlighting the possibility of inappropriate choices. A meta-transcriptome dataset with more than ninety thousand genes was also processed successfully. Conclusion: The GSV tool is a time and cost-effective tool that can be used to select reference and validation candidate genes from the biological conditions present in transcriptomic data. [ABSTRACT FROM AUTHOR]

SUMMARY: Transcriptome‐wide association studies (TWAS) can provide single gene resolution for candidate genes in plants, complementing genome‐wide association studies (GWAS) but efforts in plants have been met with, at best, mixed success. We generated expression data from 693 maize genotypes, measured in a common field experiment, sampled over a 2‐h period to minimize diurnal and environmental effects, using full‐length RNA‐seq to maximize the accurate estimation of transcript abundance. TWAS could identify roughly 10 times as many genes likely to play a role in flowering time regulation as GWAS conducted data from the same experiment. TWAS using mature leaf tissue identified known true‐positive flowering time genes known to act in the shoot apical meristem, and trait data from a new environment enabled the identification of additional flowering time genes without the need for new expression data. eQTL analysis of TWAS‐tagged genes identified at least one additional known maize flowering time gene through trans‐eQTL interactions. Collectively these results suggest the gene expression resource described here can link genes to functions across different plant phenotypes expressed in a range of tissues and scored in different experiments. Significance Statement: Large, deep and rapidly sampled RNA‐seq data produce great TWAS results, and discovering genes known to act in other tissues and using data from other experiments suggests these data can be reused to study other traits. [ABSTRACT FROM AUTHOR]

Background: The cold tolerance of rice is closely related to its production and geographic distribution. The identification of cold tolerance-related genes is of important significance for developing cold-tolerant rice. Dongxiang wild rice (Oryza rufipogon Griff.) (DXWR) is well-adapted to the cold climate of northernmost-latitude habitats ever found in the world, and is one of the most valuable rice germplasms for cold tolerance improvement. Results: Transcriptome analysis revealed genes differentially expressed between Xieqingzao B (XB; a cold sensitive variety) and 19H19 (derived from an interspecific cross between DXWR and XB) in the room temperature (RT), low temperature (LT), and recovery treatments. The results demonstrated that chloroplast genes might be involved in the regulation of cold tolerance in rice. A high-resolution SNP genetic map was constructed using 120 BC5F2 lines derived from a cross between 19H19 and XB based on the genotyping-by-sequencing (GBS) technique. Two quantitative trait loci (QTLs) for cold tolerance at the early seedling stage (CTS), qCTS12 and qCTS8, were detected. Moreover, a total of 112 candidate genes associated with cold tolerance were identified based on bulked segregant analysis sequencing (BSA-seq). These candidate genes were divided into eight functional categories, and the expression trend of candidate genes related to 'oxidation-reduction process' and 'response to stress' differed between XB and 19H19 in the RT, LT and recovery treatments. Among these candidate genes, the expression level of LOC_Os12g18729 in 19H19 (related to 'response to stress') decreased in the LT treatment but restored and enhanced during the recovery treatment whereas the expression level of LOC_Os12g18729 in XB declined during recovery treatment. Additionally, XB contained a 42-bp deletion in the third exon of LOC_Os12g18729, and the genotype of BC5F2 individuals with a survival percentage (SP) lower than 15% was consistent with that of XB. Weighted gene coexpression network analysis (WGCNA) and modular regulatory network learning with per gene information (MERLIN) algorithm revealed a gene interaction/coexpression network regulating cold tolerance in rice. In the network, differentially expressed genes (DEGs) related to 'oxidation-reduction process', 'response to stress' and 'protein phosphorylation' interacted with LOC_Os12g18729. Moreover, the knockout mutant of LOC_Os12g18729 decreased cold tolerance in early rice seedling stage signifcantly compared with that of wild type. Conclusions: In general, study of the genetic basis of cold tolerance of rice is important for the development of cold-tolerant rice varieties. In the present study, QTL mapping, BSA-seq and RNA-seq were integrated to identify two CTS QTLs qCTS8 and qCTS12. Furthermore, qRT-PCR, genotype sequencing and knockout analysis indicated that LOC_Os12g18729 could be the candidate gene of qCTS12. These results are expected to further exploration of the genetic mechanism of CTS in rice and improve cold tolerance of cultivated rice by introducing the cold tolerant genes from DXWR through marker-assisted selection. [ABSTRACT FROM AUTHOR]

Imatinib (IM), a breakthrough in chronic myeloid leukemia (CML) treatment, is accompanied by discontinuation challenges owing to drug intolerance. Although BCR-ABL1 mutation is a key cause of CML resistance, understanding mechanisms independent of BCR-ABL1 is also important. This study investigated the sphingosine-1-phosphate (S1P) signaling-associated genes (SphK1 and S1PRs) and their role in BCR-ABL1-independent resistant CML, an area currently lacking investigation. Through comprehensive transcriptomic analysis of IM-sensitive and IM-resistant CML groups, we identified the differentially expressed genes and found a notable upregulation of SphK1, S1PR2, and S1PR5 in IM-resistant CML. Functional annotation revealed their roles in critical cellular processes such as proliferation and GPCR activity. Their network analysis uncovered significant clusters, emphasizing the interconnectedness of the S1P signaling genes. Further, we identified interactors such as BIRC3, TRAF6, and SRC genes, with potential implications for IM resistance. Additionally, receiver operator characteristic curve analysis suggested these genes' potential as biomarkers for predicting IM resistance. Network pharmacology analysis identified six herbal compounds—ampelopsin, ellagic acid, colchicine, epigallocatechin-3-gallate, cucurbitacin B, and evodin—as potential drug candidates targeting the S1P signaling genes. In summary, this study contributes to efforts to better understand the molecular mechanisms underlying BCR-ABL1-independent CML resistance. Moreover, the S1P signaling genes are promising therapeutic targets and plausible new innovation avenues to combat IM resistance in cancer clinical care in the future. [ABSTRACT FROM AUTHOR]

Introduction: Expansins (EXPs) are essential components of the plant cell wall that function as relaxation factors to directly promote turgor-driven expansion of the cell wall, thereby controlling plant growth and development and diverse environmental stress responses. EXPs genes have been identified and characterized in numerous plant species, but not in sweetpotato. Results and methods: In the present study, a total of 59 EXP genes unevenly distributed across 14 of 15 chromosomes were identified in the sweetpotato genome, and segmental and tandem duplications were found to make a dominant contribution to the diversity of functions of the IbEXP family. Phylogenetic analysis showed that IbEXP members could be clustered into four subfamilies based on the EXPs from Arabidopsis and rice, and the regularity of protein motif, domain, and gene structures was consistent with this subfamily classification. Collinearity analysis between IbEXP genes and related homologous sequences in nine plants provided further phylogenetic insights into the EXP gene family. Cis-element analysis further revealed the potential roles of IbEXP genes in sweetpotato development and stress responses. RNA-seq and qRT-PCR analysis of eight selected IbEXPs genes provided evidence of their specificity in different tissues and showed that their transcripts were variously induced or suppressed under different hormone treatments (abscisic acid, salicylic acid, jasmonic acid, and 1-aminocyclopropane-1-carboxylic acid) and abiotic stresses (low and high temperature). Discussion: These results provide a foundation for further comprehensive investigation of the functions of IbEXP genes and indicate that several members of this family have potential applications as regulators to control plant development and enhance stress resistance in plants. [ABSTRACT FROM AUTHOR]

Prasiola crispa, an aerial green alga, exhibits remarkable adaptability to the extreme conditions of Antarctica by forming layered colonies capable of utilizing far-red light for photosynthesis. Despite a recent report on the structure of P. crispa's unique light-harvesting chlorophyll (Chl)-binding protein complex (Pc-frLHC), which facilitates far-red light absorption and uphill excitation energy transfer to photosystem II, the specific genes encoding the subunits of Pc-frLHC have not yet been identified. Here, we report a draft genome sequence of P. crispa strain 4113, originally isolated from soil samples on Ongul Island, Antarctica. We obtained a 92 Mbp sequence distributed in 1,045 scaffolds comprising 10,244 genes, reflecting 87.1% of the core eukaryotic gene set. Notably, 26 genes associated with the light-harvesting Chl a/b binding complex (LHC) were identified, including four Pc-frLHC genes, with similarity to a noncanonical Lhca gene with four transmembrane helices, such as Ot_Lhca6 in Ostreococcus tauri and Cr_LHCA2 in Chlamydomonas reinhardtii. A comparative analysis revealed that Pc-frLHC shares homology with certain Lhca genes found in Coccomyxa and Trebouxia species. This similarity indicates that Pc-frLHC has evolved from an ancestral Lhca gene with four transmembrane helices and branched out within the Trebouxiaceae family. Furthermore, RNAseq analysis conducted during the initiation of Pc-frLHC gene induction under red light illumination indicated that Pc-frLHC genes were induced independently from other genes associated with photosystems or LHCs. Instead, the genes of transcription factors, helicases, chaperones, heat shock proteins, and components of blue light receptors were identified to coexpress with PcfrLHC. Those kinds of information could provide insights into the expression mechanisms of Pc-frLHC and its evolutional development. [ABSTRACT FROM AUTHOR]

Background: Disease can drastically impair common bean (Phaseolus vulgaris L.) production. Anthracnose, caused by the fungal pathogen Colletotrichum lindemuthianum (Sacc. and Magnus) Briosi and Cavara, is one of the diseases that are widespread and cause serious economic loss in common bean. Results: Transcriptome analysis of the early response of common bean to anthracnose was performed using two resistant genotypes, Hongyundou and Honghuayundou, and one susceptible genotype, Jingdou. A total of 9,825 differentially expressed genes (DEGs) responding to pathogen infection and anthracnose resistance were identified by differential expression analysis. By using weighted gene coexpression network analysis (WGCNA), 2,051 DEGs were found to be associated with two resistance-related modules. Among them, 463 DEGs related to anthracnose resistance were considered resistance-related candidate genes. Nineteen candidate genes were coexpressed with three resistance genes, Phvul.001G243600, Phvul.001G243700 and Phvul.001G243800. To further identify resistance genes, 46 candidate genes were selected for experimental validation using salicylic acid (SA) and methyl jasmonate (MeJA). The results indicated that 38 candidate genes that responded to SA/MeJA treatment may be involved in anthracnose resistance in common bean. Conclusions: This study identified 38 resistance-related candidate genes involved in the early response of common bean, and 19 resistance-related candidate genes were coexpressed with anthracnose resistance genes. This study identified putative resistance genes for further resistance genetic investigation and provides an important reference for anthracnose resistance breeding in common bean. [ABSTRACT FROM AUTHOR]

SUMMARY: RNA‐Sequencing is widely used to investigate changes in gene expression at the transcription level in plants. Most plant RNA‐Seq analysis pipelines base the normalization approaches on the assumption that total transcript levels do not vary between samples. However, this assumption has not been demonstrated. In fact, many common experimental treatments and genetic alterations affect transcription efficiency or RNA stability, resulting in unequal transcript abundance. The addition of synthetic RNA controls is a simple correction that controls for variation in total mRNA levels. However, adding spike‐ins appropriately is challenging with complex plant tissue, and carefully considering how they are added is essential to their successful use. We demonstrate that adding external RNA spike‐ins as a normalization control produces differences in RNA‐Seq analysis compared to traditional normalization methods, even between two times of day in untreated plants. We illustrate the use of RNA spike‐ins with 3' RNA‐Seq and present a normalization pipeline that accounts for differences in total transcriptional levels. We evaluate the effect of normalization methods on identifying differentially expressed genes in the context of identifying the effect of the time of day on gene expression and response to chilling stress in sorghum. [ABSTRACT FROM AUTHOR]

Chronic social stress caused by repeated negative experiences in agonistic interactions induces depressive-like behavior in male mice. The aim of this study was to investigate expression changes of genes encoding proteins involved in the metabolism, receptor sensing, and transport of catecholamines, opioids, glutamate, and GABA under the influence of chronic social stress. Hypothalamus samples were analyzed using high-throughput RNA sequencing. It was shown that in the depressed animals, the expression of catecholaminergic genes Adra1b, Adrbk1, Comtd1, Ppp1r1b, Sncb, Sncg, and Th is increased, while the expression of genes Maoa and Maob is reduced. The expression of opioidergic and cannabinoidergic genes Pdyn, Penk, Pomc, Pnoc, Ogfr, and Faah was found to be upregulated, while that of genes Oprk1, Opcml, Ogfrl1, and Cnr1 was downregulated. The expression of glutamatergic genes Grik3, Grik4, Grik5, Grin1, Grm2, and Grm4 was increased, while the expression of Gria3, Grik1, Grik2, Grin2a, Grin3a, Grm5, Grm8, and Gad2 was reduced. The expression of GABAergic genes Gabre, Gabbr2, and Slc6a11 was higher, while the expression of Gabra1, Gabra2, Gabra3, Gabrb2, Gabrb3, Gabrg1, Gabrg2, and Slc6a13 was lower in the depressed animals. The results suggest that the products of genes that interact with other neurotransmitter systems (Th, Gad2, Gabra1, Gabrg2, Grin1, and Pdyn) may be of interest as potential targets for the pharmacotherapy of consequences of chronic social stress. [ABSTRACT FROM AUTHOR]

Pyramiding resistance genes may expand the efficacy and scope of a canola variety against clubroot (Plasmodiophora brassicae), a serious threat to canola production in western Canada. However, the mechanism(s) of multigenic resistance, especially the potential interaction among clubroot resistance (CR) genes, are not well understood. In this study, transcriptome was compared over three canola (Brassica napus L.) inbred/hybrid lines carrying a single CR gene in chromosome A03 (CRaM, Line 16) or A08 (Crr1rutb, Line 20), and both genes (CRaM+Crr1rutb, Line 15) inoculated with a field population (L-G2) of P. brassicae pathotype X, a new variant found in western Canada recently. The line16 was susceptible, while lines 15 and 20 were partially resistant. Functional annotation identified differential expression of genes (DEGs) involved in biosynthetic processes responsive to stress and regulation of cellular process; The Venn diagram showed that the partially resistant lines 15 and 20 shared 1,896 differentially expressed genes relative to the susceptible line 16, and many of these DEGs are involved in defense responses, activation of innate immunity, hormone biosynthesis and programmed cell death. The transcription of genes involved in Pathogen-Associated Molecular Pattern (PAMP)-Triggered and Effector-Triggered Immunity (PTI and ETI) was particularly up-regulated, and the transcription level was higher in line 15 (CRaM + Crr1rutb) than in line 20 (Crr1rutb only) for most of the DEGs. These results indicated that the partial resistance to the pathotype X was likely conferred by the CR gene Crr1rutb for both lines 15 and 20 that functioned via the activation of both PTI and ETI signaling pathways. Additionally, these two CR genes might have synergistic effects against the pathotype X, based on the higher transcription levels of defense-related DEGs expressed by inoculated line 15, highlighting the benefit of gene stacking for improved canola resistance as opposed to a single CR gene alone. [ABSTRACT FROM AUTHOR]

Background Intestinal metaplasia (IM) and intraepithelial neoplasia (IEN) are considered precursors of gastric cardia cancer (GCC). Here, we investigated the histopathologic and molecular profiles of precancerous gastric cardia lesions (PGCLs) and biomarkers for risk stratification of gastric cardia IM. Methods We conducted a hospital-based evaluation (n   =   4578) for PGCL profiles in high-incidence and non-high-incidence regions for GCC in China. We next performed 850K methylation arrays (n   =   42) and RNA-seq (n   =   44) in tissues with PGCLs. We then examined the protein expression of candidate biomarker using immunohistochemistry. Results Of the 4578 participants, 791 were diagnosed with PGCLs (600 IM, 62 IM with IEN, and 129 IEN). We found that individuals from high-incidence regions (26.7%) were more likely to develop PGCLs than those from non-high-incidence areas (13.5%). DNA methylation and gene expression alterations, indicated by differentially methylated probes (DMPs) and differentially expressed genes (DEGs), exhibited a progressive increase from type I IM (DMP = 210, DEG = 24), type II IM (DMP = 3402, DEG = 129), to type III IM (DMP = 3735, DEG = 328), peaking in IEN (DMP = 47 373, DEG = 2278). Three DEGs with aberrant promoter methylation were identified, shared exclusively by type III IM and IEN. Of these DEGs, we found that OLFM4 expression appears in IMs and increases remarkably in IENs (P  <  .001). Conclusions We highlight that type III IM and IEN share similar epigenetic and transcriptional features in gastric cardia and propose biomarkers with potential utility in risk prediction. [ABSTRACT FROM AUTHOR]

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental carcinogens accountable to developing skin cancers. Recently, we reported that exposure to benzo[a]pyrene (B[a]P), a common PAH, causes epigenetic and metabolic alterations in the initiation, promotion and progression of non-melanoma skin cancer (NMSC). As a follow-up investigation, this study examines how dietary triterpenoid ursolic acid (UA) regulates B[a]P-driven epigenetic and metabolic pathways in SKH-1 hairless mice. Our results show UA intercepts against B[a]P-induced tumorigenesis at different stages of NMSC. Epigenomic cytosines followed by guanine residues (CpG) methyl-seq data showed UA diminished B[a]P-mediated differentially methylated regions (DMRs) profiles. Transcriptomic RNA-seq revealed UA revoked B[a]P-induced differentially expressed genes (DEGs) of skin cancer-related genes, such as leucine-rich repeat LGI family member 2 (Lgi2) and kallikrein-related peptidase 13 (Klk13), indicating UA plays a vital role in B[a]P-mediated gene regulation and its potential consequences in NMSC interception. Association analysis of DEGs and DMRs found that the mRNA expression of KLK13 gene was correlated with the promoter CpG methylation status in the early-stage comparison group, indicating UA could regulate the KLK13 by modulating its promoter methylation at an early stage of NMSC. The metabolomic study showed UA alters B[a]P-regulated cancer-associated metabolisms like thiamin metabolism, ascorbate and aldarate metabolism during the initiation phase; pyruvate, citrate and thiamin metabolism during the promotion phase; and beta-alanine and pathothenate coenzyme A (CoA) biosynthesis during the late progression phase. Taken together, UA reverses B[a]P-driven epigenetic, transcriptomic and metabolic reprogramming, potentially contributing to the overall cancer interception against B[a]P-mediated NMSC. [ABSTRACT FROM AUTHOR]

Simple Summary: Eggshell gloss is an important characteristic for the manifestation of eggshell appearance. However, the reason for differences in eggshell glossiness is still unclear. The aim of this study is to perform a preliminary investigation into the formation mechanism of eggshell gloss and to identify potential genes through high-throughput sequencing. HTR1F, ZNF536, NEDD8, NGF and CALM1 were identified as potential candidate genes that may affect eggshell gloss, which provide a reference for the study of eggshell gloss and lay a foundation for improving egg glossiness in layer breeding. Eggshell gloss is an important characteristic for the manifestation of eggshell appearance. However, no study has yet identified potential candidate genes for eggshell gloss between high-gloss (HG) and low-gloss (LG) chickens. The aim of this study was to perform a preliminary investigation into the formation mechanism of eggshell gloss and to identify potential genes. The eggshell gloss of 300-day-old Rhode Island Red hens was measured from three aspects. Uterine tissues of the selected HG and LG (n = 5) hens were collected for RNA-seq. Blood samples were also collected for whole-genome resequencing (WGRS). RNA-seq analysis showed that 150 differentially expressed genes (DEGs) were identified in the uterine tissues of HG and LG hens. These DEGs were mainly enriched in the calcium signaling pathway and the neuroactive ligand–receptor interaction pathway. Importantly, these two pathways were also significantly enriched in the WGRS analysis results. Further joint analysis of WGRS and RNA-seq data revealed that 5-hydroxytryptamine receptor 1F (HTR1F), zinc finger protein 536 (ZNF536), NEDD8 ubiquitin-like modifier (NEDD8), nerve growth factor (NGF) and calmodulin 1 (CALM1) are potential candidate genes for eggshell gloss. In summary, our research provides a reference for the study of eggshell gloss and lays a foundation for improving egg glossiness in layer breeding. [ABSTRACT FROM AUTHOR]

Background: Gene network information is believed to be beneficial for disease module and pathway identification, but has not been explicitly utilized in the standard random forest (RF) algorithm for gene expression data analysis. We investigate the performance of a network-guided RF where the network information is summarized into a sampling probability of predictor variables which is further used in the construction of the RF. Results: Our simulation results suggest that network-guided RF does not provide better disease prediction than the standard RF. In terms of disease gene discovery, if disease genes form module(s), network-guided RF identifies them more accurately. In addition, when disease status is independent from genes in the given network, spurious gene selection results can occur when using network information, especially on hub genes. Our empirical analysis on two balanced microarray and RNA-Seq breast cancer datasets from The Cancer Genome Atlas (TCGA) for classification of progesterone receptor (PR) status also demonstrates that network-guided RF can identify genes from PGR-related pathways, which leads to a better connected module of identified genes. Conclusions: Gene networks can provide additional information to aid the gene expression analysis for disease module and pathway identification. But they need to be used with caution and validation on the results need to be carried out to guard against spurious gene selection. More robust approaches to incorporate such information into RF construction also warrant further study. [ABSTRACT FROM AUTHOR]

In this study, we comprehensively searched for fish‐specific genes in gnathostomes that contribute to development of the fin, a fish‐specific trait. Many previous reports suggested that animal group‐specific genes are often important for group‐specific traits. Clarifying the roles of fish‐specific genes in fin development of gnathostomes, for example, can help elucidate the mechanisms underlying the formation of this trait. We first identified 91 fish‐specific genes in gnathostomes by comparing the gene repertoire in 16 fish and 35 tetrapod species. RNA‐seq analysis narrowed down the 91 candidates to 33 genes that were expressed in the developing pectoral fin. We analyzed the functions of approximately half of the candidate genes by loss‐of‐function analysis in zebrafish. We found that some of the fish‐specific and fin development‐related genes, including fgf24 and and1/and2, play roles in fin development. In particular, the newly identified fish‐specific gene qkia is expressed in the developing fin muscle and contributes to muscle morphogenesis in the pectoral fin as well as body trunk. These results indicate that the strategy of identifying animal group‐specific genes is functional and useful. The methods applied here could be used in future studies to identify trait‐associated genes in other animal groups. [ABSTRACT FROM AUTHOR]

Eimeria media is a principal pathogen responsible for rabbit coccidiosis, targeting the rabbit’s intestinal epithelial cells. This parasitism damages the intestinal mucosal barrier, initiating a systemic immune and inflammatory response that jeopardizes the sustainable growth of rabbit farming. To understand the implications of infection on the host’s immune and metabolic responses, we employed RNA-Seq to analyze RNA from the liver and duodenum tissues of post-infected rabbits infected with both the precocious line and wild-type strain of E.media. Comprehensive transcriptomic analysis revealed that the two parasites exhibit divergent transcriptomic imprints on host tissues. While the precocious line predominantly modulates immune-centric pathways with significant differential gene enrichment, wild-type strain favors pathways that affect metabolism. In addition, our study pinpointed a set of genes that undergo significant modifications in response to these effects. These revelations grant a fresh avenue to probe deeper into the symbiotic intricacies of the E.media and its rabbit host. [ABSTRACT FROM AUTHOR]

Background: Few studies are focusing on the mechanism of erastin acts on prostate cancer (PCa) cells, and essential ferroptosis-related genes (FRGs) that can be PCa therapeutic targets are rarely known. Methods: In this study, in vitro assays were performed and RNA-sequencing was used to measure the expression of differentially expressed genes (DEGs) in erastin-induced PCa cells. A series of bioinformatic analyses were applied to analyze the pathways and DEGs. Results: Erastin inhibited the expression of SLC7A11 and cell survivability in LNCaP and PC3 cells. After treatment with erastin, the concentrations of malondialdehyde (MDA) and Fe2+ significantly increased, whereas the glutathione (GSH) and the oxidized glutathione (GSSG) significantly decreased in both cells. A total of 295 overlapping DEGs were identified under erastin exposure and significantly enriched in several pathways, including DNA replication and cell cycle. The percentage of LNCaP and PC3 cells in G1 phase was markedly increased in response to erastin treatment. For four hub FRGs, TMEFF2 was higher in PCa tissue and the expression levels of NRXN3, CLU, and UNC5B were lower in PCa tissue. The expression levels of SLC7A11 and cell survivability were inhibited after the knockdown of TMEFF2 in androgen-dependent cell lines (LNCaP and VCaP) but not in androgen-independent cell lines (PC3 and C4-2). The concentration of Fe2+ only significantly increased in TMEFF2 downregulated LNCaP and VCaP cells. Conclusion: TMEFF2 might be likely to develop into a potential ferroptosis target in PCa and this study extends our understanding of the molecular mechanism involved in erastin-affected PCa cells. [ABSTRACT FROM AUTHOR]

Pear brown rot and blossom blight caused by Monilinia laxa seriously affect pear production worldwide. Here, we compared the transcriptomic profiles of petals after inoculation with M. laxa using two pear cultivars with different levels of sensitivity to disease (Sissy, a relatively tolerant cultivar, and Kristalli, a highly susceptible cultivar). Physiological indexes were also monitored in the petals of both cultivars at 2 h and 48 h after infection (2 HAI and 48 HAI). RNA-seq data and weighted gene co-expression network analysis (WGCNA) allowed the identification of key genes and pathways involved in immune- and defenserelated responses that were specific for each cultivar in a time-dependent manner. In particular, in the Kristalli cultivar, a significant transcriptome reprogramming occurred early at 2 HAI and was accompanied either by suppression of key differentially expressed genes (DEGs) involved in the modulation of any defense responses or by activation of DEGs acting as sensitivity factors promoting susceptibility. In contrast to the considerably high number of DEGs induced early in the Kristalli cultivar, upregulation of specific DEGs involved in pathogen perception and signal transduction, biosynthesis of secondary and primary metabolism, and other defense-related responses was delayed in the Sissy cultivar, occurring at 48 HAI. The WGCNA highlighted one module that was significantly and highly correlated to the relatively tolerant cultivar. Six hub genes were identified within this module, including three WRKY transcription factor-encoding genes: WRKY 65 (pycom05g27470), WRKY 71 (pycom10g22220), and WRKY28 (pycom17g13130), which may play a crucial role in enhancing the tolerance of pear petals to M. laxa. Our results will provide insights into the interplay of the molecular mechanisms underlying immune responses of petals at the pear–M. laxa pathosystem. [ABSTRACT FROM AUTHOR]

Enterococcus faecalis, a formidable nosocomial and community-acquired opportunistic pathogen, can persist a wide range of extreme environments, including low pH and nutrient deficiency. Clarifying the survival mechanism of E. faecalis in low-pH conditions is the key to combating the infectious diseases caused by E. faecalis. In this study, we combined transcriptome profiling (RNA-seq) and transposon insertion sequencing (TIS) to comprehensively understand the genes that confer these features on E. faecalis. The metadata showed that genes whose products are involved in cation transportation and amino acid biosynthesis were predominantly differentially expressed under acid conditions. The products of genes such as opp1C and copY reduced the hydrion concentration in the cell, whereas those of gldA2, gnd2, ubiD, and ubiD2 mainly participated in amino metabolism, increasing matters to neutralize excess acid. These, together with the folE and hexB genes, which are involved in mismatch repair, form a network of E. faecalis genes necessary for its survival under acid conditions. [ABSTRACT FROM AUTHOR]

Introduction: Branching angle is an essential trait in determining the planting density of rapeseed (Brassica napus L.) and hence the yield per unit area. However, the mechanism of branching angle formation in rapeseed is not well understood. Methods: In this study, two rapeseed germplasm with extreme branching angles were used to construct an F2 segregating population; then bulked segregant analysis sequencing (BSA-seq) and quantitative trait loci (QTL) mapping were utilized to localize branching anglerelated loci and combined with transcriptome sequencing (RNA-seq) and quantitative real-time PCR (qPCR) for candidate gene mining. Results and discussion: A branching angle-associated quantitative trait loci (QTL) was mapped on chromosome C3 (C3: 1.54-2.65 Mb) by combining BSAseq as well as traditional QTL mapping. A total of 54 genes had SNP/Indel variants within the QTL interval were identified. Further, RNA-seq of the two parents revealed that 12 of the 54 genes were differentially expressed between the two parents. Finally, we further validated the differentially expressed genes using qPCR and found that six of them presented consistent differential expression in all small branching angle samples and large branching angles, and thus were considered as candidate genes related to branching angles in rapeseed. Our results introduce new candidate genes for the regulation of branching angle formation in rapeseed, and provide an important reference for the subsequent exploration of its formation mechanism. [ABSTRACT FROM AUTHOR]

Yellow catfish (Pelteobagrus fulvidraco) is an important freshwater fish species in aquaculture. However, as intensive farming has rapidly expanded, infectious diseases caused by various bacteria, such as Aeromonas hydrophila, have also increased. We conducted a transcriptomic analysis of head kidney from P. fulvidraco stimulated by A. hydrophila strain SHOU. A total of 43,249 unigenes with an average length of 1342 bp were obtained following assembly and annotation. By analyzing GO and KEGG enrichment, many differentially expressed genes (DEGs) and pathways related to immunity were identified. Out of the 973 DEGs that were identified, 553 were upregulated and 420 were downregulated. Moreover, KEGG enrichment analysis revealed that the innate immune system, including the TNF signaling pathway, NF-kappa B signaling pathway, and the Toll-like receptor signaling pathway involved in the defense, is activated against infectious response. Real-time quantitative reverse transcription-PCR (qRT-PCR) analysis demonstrated that immune response genes were upregulated in response to A. hydrophila stimulation compared to the control. In conclusion, this study provides valuable insights into the immune defense mechanism of P. fulvidraco and sheds light on the host immune genes involved in the response to bacterial infection. [ABSTRACT FROM AUTHOR]

Long-read transcriptome sequencing provides us with a convenient tool for the thorough study of biological processes such as neuronal plasticity. Here, we aimed to perform transcriptional profiling of rat hippocampal primary neuron cultures after stimulation with picrotoxin (PTX) to further understand molecular mechanisms of neuronal activation. To overcome the limitations of short-read RNA-Seq approaches, we performed an Oxford Nanopore Technologies MinION-based long-read sequencing and transcriptome assembly of rat primary hippocampal culture mRNA at three time points after the PTX activation. We used a specific approach to exclude uncapped mRNAs during sample preparation. Overall, we found 23,652 novel transcripts in comparison to reference annotations, out of which ~6000 were entirely novel and mostly transposon-derived loci. Analysis of differentially expressed genes (DEG) showed that 3046 genes were differentially expressed, of which 2037 were upregulated and 1009 were downregulated at 30 min after the PTX application, with only 446 and 13 genes differentially expressed at 1 h and 5 h time points, respectively. Most notably, multiple genes encoding ribosomal proteins, with a high basal expression level, were downregulated after 30 min incubation with PTX; we suggest that this indicates redistribution of transcriptional resources towards activity-induced genes. Novel loci and isoforms observed in this study may help us further understand the functional mRNA repertoire in neuronal plasticity processes. Together with other NGS techniques, differential gene expression analysis of sequencing data obtained using MinION platform might provide a simple method to optimize further study of neuronal plasticity. [ABSTRACT FROM AUTHOR]

High temperatures are increasingly becoming a prominent environmental factor accelerating the adverse influence on the growth and development of maize (Zea mays L.). Therefore, it is critical to identify the key genes and pathways related to heat stress (HS) tolerance in maize. Great challenges have been faced in dissecting genetic mechanisms and uncovering master genes for HS tolerance. Here, Z58D showed more thermotolerance than AF171 at the seedling stage with a lower wilted leaf rate and H2O2 accumulation under HS conditions. Transcriptomic analysis identified 3006 differentially expressed genes (DEGs) in AF171 and 4273 DEGs in Z58D under HS treatments, respectively. Subsequently, GO enrichment analysis showed that commonly upregulated genes in AF171 and Z58D were significantly enriched in the following biological processes, including protein folding, response to heat, response to temperature stimulus and response to hydrogen peroxide. Moreover, the comparison between the two inbred lines under HS showed that response to heat and response to temperature stimulus were significantly over-represented for the 1234 upregulated genes in Z58D. Furthermore, more commonly upregulated genes exhibited higher expression levels in Z58D than AF171. In addition, maize inbred CIMBL55 was verified to be more tolerant than B73, and more commonly upregulated genes also showed higher expression levels in CIMBL55 than B73 under HS. These consistent results indicate that heat-resistant inbred lines may coordinate the remarkable expression of genes in order to recover from HS. Additionally, 35 DEGs were conserved among five inbred lines via comparative transcriptomic analysis. Most of them were more pronounced in Z58D than AF171 at the expression levels. These candidate genes may confer thermotolerance in maize. [ABSTRACT FROM AUTHOR]

Soybean [Glycine max (L.) Merr.] isoflavones, which are secondary metabolites with various functions, are included in food, cosmetics, and medicine. However, the molecular mechanisms regulating the glycosylation and malonylation of isoflavone glycoconjugates remain unclear. In this study, we conducted an RNA-seq analysis to compare soybean genotypes with different isoflavone contents, including Danbaek and Hwanggeum (low-isoflavone cultivars) as well as DB-088 (high-isoflavone mutant). The transcriptome analysis yielded over 278 million clean reads, representing 39,156 transcripts. The analysis of differentially expressed genes (DEGs) detected 2654 up-regulated and 1805 down-regulated genes between the low- and high-isoflavone genotypes. The putative functions of these 4459 DEGs were annotated on the basis of GO and KEGG pathway enrichment analyses. These DEGs were further analyzed to compare the expression patterns of the genes involved in the biosynthesis of secondary metabolites and the genes encoding transcription factors. The examination of the relative expression levels of 70 isoflavone biosynthetic genes revealed the HID, IFS, UGT, and MAT expression levels were significantly up/down-regulated depending on the genotype and seed developmental stage. These expression patterns were confirmed by quantitative real-time PCR. Moreover, a gene co-expression analysis detected potential protein–protein interactions, suggestive of common functions. The study findings provide valuable insights into the structural genes responsible for isoflavone biosynthesis and accumulation in soybean seeds. [ABSTRACT FROM AUTHOR]

Although gene/genome duplications in the early stage of vertebrates have been thought to provide major resources of raw genetic materials for evolutionary innovations, it is unclear whether they continuously contribute to the evolution of morphological complexity during the course of vertebrate evolution, such as the evolution from two heart chambers (fishes) to four heart chambers (mammals and birds). We addressed this issue by our heart RNA‐Seq experiments combined with published data, using 13 vertebrates and one invertebrate (sea squirt, as an outgroup). Our evolutionary transcriptome analysis showed that number of ancient paralogous genes expressed in heart tends to increase with the increase of heart chamber number along the vertebrate phylogeny, in spite that most of them were duplicated at the time near to the origin of vertebrates or even more ancient. Moreover, those paralogs expressed in heart exert considerably different functions from heart‐expressed singletons: the former are functionally enriched in cardiac muscle and muscle contraction‐related categories, whereas the latter play more basic functions of energy generation like aerobic respiration. These findings together support the notion that recruiting anciently paralogous genes that are expressed in heart is associated with the increase of chamber number in vertebrate evolution. Research Highlights: Correlation between the expression of paralogous genes and heart chamber complexity.Paralogs expressed in heart exert considerably different functions from heart‐expressed singletons.Paralogous genes expressed in heart is associated with the increase of chamber number in evolution. [ABSTRACT FROM AUTHOR]

Soybean (Glycine max) is an economically important cash crop and food source that serves as a key source of high-quality plant-derived protein and oil. Seed vigor is an important trait that influences the growth and development of soybean plants in an agricultural setting, underscoring a need for research focused on identifying seed vigor-related genetic loci and candidate genes. In this study, a population consisting of 207 chromosome segment substitution lines (CSSLs) derived from the crossing and continuous backcrossing of the Suinong14 (improved cultivar, recurrent parent) and ZYD00006 (wild soybean, donor parent) soybean varieties was leveraged to identify quantitative trait loci (QTLs) related to seed vigor. The candidate genes detected using this approach were then validated through RNA-seq, whole-genome resequencing, and qPCR approaches, while the relationship between specific haplotypes and seed vigor was evaluated through haplotype analyses of candidate genes. Phenotypic characterization revealed that the seed vigor of Suinong14 was superior to that of ZYD00006, and 20 total QTLs were identified using the selected CSSLs. Glyma.03G256700 was also established as a seed vigor-related gene that was upregulated in high-vigor seeds during germination, with haplotypes for this candidate gene also remaining consistent with observed soybean seed vigor. The QTLs identified herein can serve as a foundation for future marker-assisted and convergent breeding efforts aimed at improving seed vigor. In addition, future molecular and functional research focused on Glyma.03G256700 has the potential to elucidate the signaling network and key regulatory mechanisms that govern seed germination in soybean plants. [ABSTRACT FROM AUTHOR]

Heat stress, as a negative factor, severely threatens the quality and production of bottle gourd, which prefers to grow in a warm environment. To understand which genes are involved in the resistance to heat stress in bottle gourd (Lagenaria siceraria (Mol.) Standl.), we analyzed the characteristics of two genetic bottle gourd varieties, "Mei feng"-MF (heat resistant) and "Lv long"-LL (heat sensitive). Under heat stress, MF plants exhibited a higher survival rate, lower relative electrolytic leakage, and decreased stomatal aperture compared with LL. In addition, RNA-Seq was carried out on the two varieties under normal conditions and heat stress. The results revealed a total of 1485 up-regulated and 946 down-regulated genes under normal conditions, while 602 genes were up-regulated and 1212 genes were down-regulated under heat stress. Among these genes, several differentially expressed genes (DEGs) involved in the MAPK (mitogen-activated protein kinase) signaling pathway and members of bHLH (basic helix-loop-helix) transcription factors showed significant up- or down-regulation after heat stress. Next, to validate these findings, we conducted quantitative real-time PCR (qRT-PCR) analysis, which confirmed the expression patterns of the genes detected through RNA-Seq. Collectively, the DEGs between the two contrasting cultivars identified in our study provide novel insight into excavating helpful candidate genes associated with heat tolerance in bottle gourd. [ABSTRACT FROM AUTHOR]

Objectives: Gain insights into the pathophysiology of idiopathic subglottic stenosis (iSGS) by investigating differences in transcriptome of subglottic mucosal tissue between patients with iSGS and controls, and between tracheal and subglottic tissue within patients. Methods: RNA sequencing was conducted on biopsied mucosal samples collected from subglottic and tracheal (in-patient control) regions in iSGS patients, and from subglottis in controls. The gene expression differences were validated on a protein level by (1) staining the tissue samples obtained from a second cohort of patients and controls; and (2) in vitro functional assays using primary subglottic epithelial cells from both iSGS patients and healthy donors. Results: We found 7 upregulated genes in the subglottic region of iSGS patients relative to both the tracheal mucosa and subglottic region of controls. A gene ontology enrichment analysis found that the epithelial cell differentiation and cornification pathways are significant, involving specifically 3 of the genes: involucrin (IVL), small proline rich protein 1B (SPRR1B), and keratin 16 (KRT16). Involvement of these pathways suggests squamous metaplasia of the epithelium. Histological analyses of epithelium in subglottic mucosal biopsies revealed squamous metaplasia in 41% of the samples from iSGS patients and in 25% from controls. Immunohistochemical evaluation of the samples presented with squamous epithelium revealed increased expression of the protein encoded by SPRR1B, hyperproliferative basal cells, shedding of apical layers, and accompanying lesions in iSGS compared to CTRL. Cultured primary subglottic epithelial cells from iSGS patients had higher proliferation rates compared to healthy donors and squamous metaplastic differentiation formed thinner epithelia with increased expression proteins encoded by INV, SPRR1B, and KRT16, suggesting intrinsic dysfunction of basal cells in iSGS. Conclusions: Abnormal squamous differentiation of epithelial cells may contribute to the pathogenesis of iSGS. Patients having metaplastic epithelial phenotype may be sensitive to drugs that reverse it to a normal phenotype. [ABSTRACT FROM AUTHOR]

Diabetic nephropathy(DN) remains a significant risk factor for cardiovascular and all-cause mortality, and end-stage renal disease (ESRD) associated with it is growing in prevalence.However, there is absolutely no curative strategy for DN. We subjected db/db and control mouse kidneys to transcriptional sequencing analysis to obtain transcriptome expression profile data in the diabetic nephropathy.We next performed differential analysis of db/db and control mice kidney sequencing data to obtain differentially expressed genes. The differential expressed genes were intersected with the oxidative stress and inflammatory response related genes derived from the MGI/MsiDB gene set to yield oxidative stress inflammatory response related differential 122 genes (OIRDEGs). To further clarify the biological functions of DEGs, we conducted GOKEGG analysis and obtained the top 20 genes by five computational algorithms of the cytohubba plugin via cytoscape, respectively. The genes obtained by the five algorithms were intersected and the intersection genes were considered as key genes,including Cd40lg, Il2rb, Lck, Il2rg, Zap70, Serpinb1a. Also,we used GSEA and immune infiltration analysis to clarify the biological signaling pathways and immune cell infiltration that are substantial in the diabetic nephropathy.Correlation studies of key genes with immune cell infiltration revealed that they were correlated with the majority types of T cells while only with two types of B cells.Then, we predicted miRNA and TF for the key genes and constructed the interaction network. Finally, the expression differences of key genes were examined by validation dataset and RT-PCR experiment.In conclusion,we have identified key genes associated with T cell immune response in a diabetic nephropathy model, which bear significance in the etiopathogenesis of immunological injury in diabetic nephropathy and provide an innovative proposal for the recognition and management of DN. • Diabetic nephropathy is a major cause of end-stage renal disease. • Pathogenesis of diabetic nephropathy is undefined and key genes have essential contributions. • Key genes in diabetic nephropathy are associated with T cell development and signaling pathways. • T cell-associated immune-inflammatory responses may be a novel target for intervention in diabetic nephropathy. [ABSTRACT FROM AUTHOR]

Despite being a non-hematophagous leech, Whitmania pigra is widely used in traditional Chinese medicine for the treatment of antithrombotic diseases. In this study, we provide a high quality genome of W. pigra and based on which, we performed a systematic identification of the potential antithrombotic genes and their corresponding proteins. We identified twenty antithrombotic gene families including thirteen coagulation inhibitors, three platelet aggregation inhibitors, three fibrinolysis enhancers, and one tissue penetration enhancer. Unexpectedly, a total of 79 antithrombotic genes were identified, more than a typical blood-feeding Hirudinaria manillensis, which had only 72 antithrombotic genes. In addition, combining with the RNA-seq data of W. pigra and H. manillensis, we calculated the expression levels of antithrombotic genes of the two species. Five and four gene families had significantly higher and lower expression levels in W. pigra than in H. manillensis, respectively. These results showed that the number and expression level of antithrombotic genes of a non-hematophagous leech are not always less than those of a hematophagous leech. Our study provides the most comprehensive collection of antithrombotic biomacromolecules from a non-hematophagous leech to date and will significantly enhance the investigation and utilization of leech derivatives in thrombosis therapy research and pharmaceutical applications. [ABSTRACT FROM AUTHOR]

Simple Summary: The reproductive performance of pigs can vary due to the influence of genetic factors, and this is aggravated by environmental factors. Multiple complex factors affect the difference in the reproductive performance of boars in adulthood. This study examined those genetic differences in young piglets, at a time when they are less affected by the environment. Testicular tissue samples were used to explore candidate genes and related pathways affecting testicular size during early development. This experiment into the reproductive performance of pig breeds using the RNA-Seq analysis of testicular tissue with early developmental differences can provide a theoretical basis for breeding by determining key candidate genes that control testicular predevelopment. The data will be used for subsequent research into the genetic mechanism of boars, which is important for early breeding and improvement. The RNA-Seq technology was used to screen the key genes that affect the early development of the testes of Duroc × Landrace × Yorkshire piglets, to determine the regulatory pathway and provide reference for subsequent reproductive performance research, breeding, and other production practices. This study selected 14-day-old Duroc × Landrace × Yorkshire piglets as the trial animals. Testes from piglets with similar weights and no pathological changes were divided into small testis (ST) and large testis (LT) groups, and the RNA-Seq screening of differentially expressed genes (DEGs) was performed to find candidate genes and regulatory pathways related to early testicular development. The results show that 570 DEGs were found in the ST and LT groups, with 281 upregulated and 289 downregulated. The DEGs were mainly enriched on 47 gene ontology (GO) functional items. The Kyoto encyclopedia of genes and genotypes (KEGG) enrichment analysis found that there were 44 significantly enriched KEGG signal pathways, and the regulation of testicular development mainly focused on the arachidonic acid metabolism, Wnt signaling pathway and GnRH secretion pathways. The PTGES, SFRP1, SPP1, PLA2G4E, KCNJ5, PTGS2, and HCN1 genes were found to be as closely related to the testicular development of these Duroc × Landrace × Yorkshire piglets, and the differential gene expression was consistent with the real-time quantitative reverse transcription PCR (real-time qRT-PCR) validation results. This study was validated by high-throughput sequencing analysis and real-time qRT-PCR, and showed that the PTGES, SFRP1, SPP1, PLA2G4E, KCNJ5, PTGS2, and HCN1 genes may be involved in the regulation of germ cell development, spermatogenesis and semen traits. These should be further studied as candidate genes for early testicular development and reproductive trait regulation in boars. [ABSTRACT FROM AUTHOR]

Euonymus japonicus is one of the most low-temperature-tolerant evergreen broad-leaved tree species in the world and is widely used in urban greening. However, there are very few molecular biology studies on its low-temperature tolerance mechanism. So far, no researcher has selected and reported on its reference genes. In this study, 21 candidate reference genes (12 traditional housekeeping genes and 9 other genes) were initially selected based on gene expression and coefficient of variation (CV) through RNA-Seq (unpublished data), and qRT-PCR was used to detect the expression levels of candidate reference genes in three different groups of samples (leaves under different temperature stresses, leaves of plants at different growth stages, and different organs). After further evaluating the expression stability of these genes using geNorm, NormFinder, Bestkeeper, and RefFind, the results show that the traditional housekeeping gene eIF5A and the new reference gene RTNLB1 have good stability in the three different groups of samples, so they are reference genes with universality. In addition, we used eIF5A and RTNLB1 as reference genes to calibrate the expression pattern of the target gene EjMAH1, which confirmed this view. This article is the first to select and report on the reference gene of E. japonicus, laying the foundation for its low-temperature tolerance mechanism and other molecular biology research. [ABSTRACT FROM AUTHOR]

Maize has become one of the most widely grown grains in the world, and the stay-green mutant allows these plants to maintain their green leaves and photosynthetic potential for longer following anthesis than in non-mutated plants. As a result, stay-green plants have a higher production rate than non-stay-green varieties due to their prolonged grain-filling period. In this study, the candidate genes related to the visual stay-green at the maturation stage of maize were investigated. The F2 population was derived from the T01 (stay-green) and the Xin3 (non-stay-green) cross. Two bulked segregant analysis pools were constructed. According to the method of combining ED (Euclidean distance), Ridit (relative to an identified distribution unit), SmoothG, and SNP algorithms, a region containing 778 genes on chromosome 9 was recognized as the candidate region associated with the visual stay-green in maize. A total of eight modules were identified using WGCNA (weighted correlation network analysis), of which green, brown, pink, and salmon modules were significantly correlated with visual stay-green. BSA, combined with the annotation function, discovered 7 potential candidate genes, while WGCNA discovered 11 stay-green potential candidate genes. The candidate range was further reduced due through association analysis of BSA-seq and RNA-seq. We identified Zm00001eb378880, Zm00001eb383680, and Zm00001eb384100 to be the most likely candidate genes. Our results provide valuable insights into this new germplasm resource with reference to increasing the yield for maize. [ABSTRACT FROM AUTHOR]

Objective: This study aimed to evaluate the microbial functional profile of biofilms related to caries-free (CF, n = 6) and caries-arrested (CI, n = 3) compared to caries-active (CA, n = 5) individuals. Materials and methods: A metatranscriptomic was performed in supragingival biofilm from different clinical conditions related to caries or health. Total RNA was extracted and cDNAs were obtained and sequenced (Illumina HiSeq3000). Trimmed data (SortMeRNA) were submitted to the SqueezeMeta pipeline in the co-assembly mode for functional analysis and further differential gene expression analysis (DESeq2) and weighted gene co-expression network analysis (WCGNA) to explore and identify gene modules related to these clinical conditions. Results: A total of 5303 genes were found in the metatranscriptomic analysis. A co-expression network identified the most relevant modules strongly related to specific caries status. Correlation coefficients were calculated between the eigengene modules and the clinical conditions (CA, CI, and CF) discriminating multiple modules. CA and CI showed weak correlation coefficient strength across the modules, while the CF condition presented a very strong positive correlation coefficient (r = 0.9, p value = 4 × 10−9). Pearson's test was applied to further analyze the module membership and gene significance in CF conditions, and the most relevant were HSPA1s-K03283, Epr- K13277, and SLC1A-K05613. Gene Ontology (GO) shows important bioprocesses, such as two-component system, fructose and mannose metabolism, pentose and glucuronate interconversions, and flagellar assembly (p-adjust < 0.05). The ability to use different carbohydrates, integrate multiple signals, swarm, and bacteriocin production are significant metabolic advantages in the oral environment related to CF. Conclusions: A distinct functional health profile could be found in CF, where co-occurring genes can act in different pathways at the same time. Genes HSPA1s, Epr, and SLC1A may be appointed as potential biomarkers for caries-free biofilms. Clinical relevance: Potential biomarkers for caries-free biofilms could contribute to the knowledge of caries prevention and control. [ABSTRACT FROM AUTHOR]

Oil palm (Elaeis guineensis) is the most important tropical oil-bearing crop species worldwide. MADS-box proteins, which play crucial roles in plant growth and development and are involved in various physiological and biochemical processes, compose one of the largest families of plant transcription factors. In this study, 42 MADS-box genes were screened from the mesocarp transcriptome database of oil palm fruit, and their phylogenetic relationships with Arabidopsis thaliana MADS-box genes were analyzed. Based on the results, MADS-box genes from oil palm mesocarp were classified into four groups: MIKCc-type, MIKC*-type, Mα-type, and Mγ-type MADS-box genes. Members of the subfamilies were classified according to the presence of three specific protein motifs. To explore the differential expression of the MADS-box genes, the dynamic expression of all selected MADS-box genes in oil palm was measured by RNA-seq. The high expression of specific MADS-box genes in the mesocarp of oil palm during different developmental stages indicates that those genes may play important roles in the cell division of and metabolite accumulation in the fruit and could become important targets for fruit development and oil accumulation research in oil palm. [ABSTRACT FROM AUTHOR]

Background Idiopathic pulmonary fibrosis (IPF) is increasingly recognized as a chronic, progressive and fatal lung disease with an unknown etiology. Current studies focus on revealing the genetic factors in the risk of IPF, making the integrative analysis of genetic variations and transcriptomic alterations of substantial value. Aim This study aimed to improve the understanding of the molecular basis of IPF through an integrative analysis of whole-exome sequencing (WES), bulk RNA sequencing (RNA-seq) and single-cell RNA sequencing (scRNA-seq) data. Methods WES is a powerful tool for studying the genetic basis of IPF, allowing for the identification of genetic variants that may be associated with the development of the disease. RNA-seq data provide a comprehensive view of the transcriptional changes in IPF patients, while scRNA-seq data offer a more granule view of cell-type-specific alterations. Results In this study, we identified a comprehensive mutational landscape of recurrent genomic and transcriptomic variations, including single-nucleotide polymorphisms, CNVs and differentially expressed genes, in IPF populations, which may play a significant role in the development and progression of IPF. Conclusions Our study provided valuable insights into the genetic and transcriptomic variations associated with IPF, revealing changes in gene expression that may contribute to disease development and progression. These findings highlight the importance of an integrative approach to understanding the molecular mechanisms underlying IPF and may pave the way for identifying potential therapeutic targets. [ABSTRACT FROM AUTHOR]

Cancer, a genetic disease, is considered one of the leading causes of death globally and affects people of all ages. Ribonucleic acid sequencing (RNA-Seq) is a technique used to quantify the expression of genes of interest and can be used to classify cancer tumor types. This paper describes a machine learning technique to classify cancer tissue samples by tumor type, such as breast cancer, lung cancer, colon cancer, and others. More than 60,000 RNA-Seq features were analyzed using six different machine learning classification algorithms, both individually and as an ensemble. Numerous dimensionality reduction techniques addressed the challenges of working with enormous amounts of genetic data. In particular, we were able to reduce the number of features from over 60,000 to 660 in the random forest feature selection and to 68 factor features using factor analysis with an accuracy of 99% in classifying tumor types. [ABSTRACT FROM AUTHOR]

Accurate and standardized quantification of reverse transcription PCR (qRT-PCR) results relies on the use of a dependable reference gene. The precise control of transgene expression in terms of both spatial and temporal aspects necessitates the utilization of tissue-specific gene promoters. However, the identification of stable reference genes across various tissues, particularly in fruits at different ripening stages, as well as tissue-specific genes in bitter gourds, remains largely unexplored. In this study, we employed RNA-Seq-based transcriptome datasets obtained from nine tissues to comprehensively screen for new reference genes (NRGs) and tissue-specific genes. Through the utilization of five algorithms in conjunction with qRT-PCR analysis, we successfully identified two highly stable reference genes, namely HMG1/2 and PHOS32, from a pool of 11 NRGs and five traditional reference genes (TRGs). To validate their reliability, we performed expression pattern analysis of two genes associated with fruit ripening (McACO1 and McACO2) using HMG1/2 and PHOS32, as well as an unstable reference gene, HSCP2. Furthermore, we conducted qRT-PCR validation of 12 tissue-specific genes using HMG1/2 as the reference gene. This study not only contributes to the precise normalization of target genes in bitter gourd but also provides a solid foundation for regulating transgenes through the utilization of suitable tissue-specific promoters. [ABSTRACT FROM AUTHOR]

Background. The mitochondrial genomes (mitogenomes) of metazoans generally include the same set of protein-coding genes, which ensures the homology of mitochondrial genes between species. The mitochondrial genes are often used as reference data for species identification based on genetic data (DNA barcoding). The need for such reference data has been increasing due to the application of environmental DNA (eDNA) analysis for environmental assessments. Recently, the number of publicly available sequence reads obtained with next-generation sequencing (NGS) has been increasing in the public database (the NCBI Sequence Read Archive, SRA). Such freely available NGS reads would be promising sources for assembling mitochondrial proteincoding genes (mPCGs) of organisms whose mitochondrial genes are not available in GenBank. The present study aimed to assemble annelid mPCGs from raw data deposited in the SRA. Methods. The recent progress in the classification of Annelida was briefly introduced. In the present study, the mPCGs of 32 annelid species of 19 families in clitellates and allies in Sedentaria (echiurans and polychaetes) were newly assembled from the reads deposited in the SRA. Assembly was performed with a recently published pipeline mitoRNA, which includes cycles of Bowtie2 mapping and Trinity assembly. Assembled mPCGs were deposited in GenBank as Third Party Data (TPA) data. A phylogenetic tree was reconstructed with maximum likelihood (ML) analysis, together with other mPCGs deposited in GenBank. Results and Discussion. mPCG assembly was largely successful except for Travisia forbesii; only four genes were detected from the assembled contigs of the species probably due to the reads targeting its parasite. Most genes were largely successfully obtained, whereas atp8, nad2, and nad4l were only successful in 22-24 species. The high nucleotide substitution rates of these genes might be relevant to the failure in the assembly although nad6, which showed a similarly high substitution rate, was successfully assembled. Although the phylogenetic positions of several lineages were not resolved in the present study, the phylogenetic relationships of some polychaetes and leeches that were not inferred by transcriptomes were well resolved probably due to a more dense taxon sampling than previous phylogenetic analyses based on transcriptomes. Although NGS data are generally better sources for resolving phylogenetic relationships of both higher and lower classifications, there are ensuring needs for specific loci of the mitochondrial genes for analyses that do not require high resolutions, such as DNA barcoding, eDNA, and phylogenetic analysis among lower taxa. Assembly from publicly available NGS reads would help design specific primers for the mitochondrial gene sequences of species, whose mitochondrial genes are hard to amplify by Sanger sequencing using universal primers. [ABSTRACT FROM AUTHOR]

For more than 100 years, the fruit fly Drosophila melanogaster has been one of the most studied model organisms. Here, we present a single-cell atlas of the adult fly, Tabula Drosophilae, that includes 580,000 nuclei from 15 individually dissected sexed tissues as well as the entire head and body, annotated to >250 distinct cell types. We provide an in-depth analysis of cell type-related gene signatures and transcription factor markers, as well as sexual dimorphism, across the whole animal. Analysis of common cell types between tissues, such as blood and muscle cells, reveals rare cell types and tissue-specific subtypes. This atlas provides a valuable resource for the Drosophila community and serves as a reference to study genetic perturbations and disease models at single-cell resolution.

n-Caproic acid (CA) is gaining increased attention due to its high value as a chemical feedstock. Ruminococcaceae bacterium strain CPB6 is an anaerobic mesophilic bacterium that is highly prolific in its ability to perform chain elongation of lactate to CA. However, little is known about the genome-wide transcriptional analysis of strain CPB6 for CA production triggered by the supplementation of exogenous lactate. In this study, cultivation of strain CPB6 was carried out in the absence and presence of lactate. Transcriptional profiles were analyzed using RNA-seq, and differentially expressed genes (DEGs) between the lactate-supplemented cells and control cells without lactate were analyzed. The results showed that lactate supplementation led to earlier CA p,roduction, and higher final CA titer and productivity. 295 genes were substrate and/or growth dependent, and these genes cover crucial functional categories. Specifically, 5 genes responsible for the reverse β-oxidation pathway, 11 genes encoding ATP-binding cassette (ABC) transporters, 6 genes encoding substrate-binding protein (SBP), and 4 genes encoding phosphotransferase system (PTS) transporters were strikingly upregulated in response to the addition of lactate. These genes would be candidates for future studies aiming at understanding the regulatory mechanism of lactate conversion into CA, as well as for the improvement of CA production in strain CPB6. The findings presented herein reveal unique insights into the biomolecular effect of lactate on CA production at the transcriptional level.

Cases of Leber congenital amaurosis caused by mutations in CRX (LCA7) exhibit an early form of the disease and show signs of significant photoreceptor dysfunction and eventual loss. To establish a translational in vitro model system to study gene-editing-based therapies, we generated LCA7 retinal organoids harboring a dominant disease-causing mutation in CRX. Our LCA7 retinal organoids develop signs of immature and dysfunctional photoreceptor cells, providing us with a reliable in vitro model to recapitulate LCA7. Furthermore, we performed a proof-of-concept study in which we utilize allele-specific CRISPR/Cas9-based gene editing to knock out mutant CRX and saw moderate rescue of photoreceptor phenotypes in our organoids. This work provides early evidence for an effective approach to treat LCA7, which can be applied more broadly to other dominant genetic diseases.